This is a Fast-Track proposal to develop a multispectral optical tomography small-animal imaging system for use by the biomedical and pharmaceutical research communities. It will combine the experience CRI has acquired in multispectral, planar small-animal imaging (through its development of the Maestro(tm) system) with novel hyperspectral optical tomography algorithms and will be realized using a unique optical design. The primary aim of this proposal is to refine 3-dimensional (3D) localization of point and extended fluorescent sources within small animals. Tomographic approaches are essential for achieving truly quantitative, depth- resolved imaging. Partners in this project include leaders in optical design, algorithmic development, dye chemistry, advanced small-animal models of disease, and directors of animal imaging facilities. Key properties include: 1) the use of a spectrally tunable filter with unmatched flexibility for multiplexed fluorophore signal detection in the presence of significant autofluorescence;2) a novel optic that enables multi- view detection;3) a customized multispectral fluorescent label strategy;and 4) no requirement for camera or subject movement, compression of the subject between glass plates, or submersion of the subject in index- and scatter-matching fluids. Phase I will accomplish: construction of a first-generation instrument with off-the-shelf optics;radiometric characterization;surface profilometry, and confirmation via simulation that the instrument will accomplish 3D reconstructions with a resolution of 2 millimeters or better. Phase II will be dedicated to building a second- generation version of the instrument with custom, high-efficiency optics, and will add a variety of important algorithmic, computational and reagent components for optimal performance, with an emphasis on quantitative validation. The system will be deployed at several animal imaging facilities. A variety of animal models of disease, including metastatic breast cancer, pulmonary, cardiac and vascular disease will be studied and the performance of the Maestro-3D compared to competing optical and non-optical imaging modalities. In addition, CRI will work on methods to accelerate time consuming tomographic reconstructions. Finally, easy-to-use software for control, display, quantitation and integration into laboratory information systems will be developed.